> In article <2oejvv$scv at fermat.mayo.edu>, volcs at fermat.mayo.edu (Sam
> Volchenboum) wrote:
> > I need to precipitate a protein sample coming off a FPLC column for
> > subsequent SDS-PAGE. I was going to precipitate with 10% TCA and then wash
> > with 10% TCA, but I read that the TCA can change the pH so as to affect the
> > PAGE. Then someone told me to wash with acetone. Fine, but can't I just
> > precipitate with one volume of acetone from the start? Anyways, do any of
> > you have ideas/protocols/refs which will precipitate small amounts of
> > protein and not affect the PAGE? (Or should I just use TCA and be done with
> > it...)
> TCA or TCA/deoxycholate pption and then re-pHing with saturated un-pHed
> Tris, once your sample is in loading buffer, so that the BPB is blue again
> is one that I use often. However, it is easy to over pH your sample.
> Another is to ppte your proteins with 5-10X vol acetone -I find 5 vol is
> usually good enough but you can use 10 vol to be sure. A third way is, in
> fact, a method for removal of lipids:
> Add 0.4 ml of methanol to 0.1 ml of protein soln. Vortex and add 0.1 ml
> chloroform, vortex and centrifuge 1 min (microcentrifuge). Add 0.3 ml
> water, vortex and centrifuge 1 min. Remove upper layer and discard. Add 0.3
> ml methanol to the lower chloroform phase, mix and centrifuge 2 min. Remove
> Better late than never.
> John McDougall / TEL: 47-776-44479
> NFH / FAX: 47-776-71832
> Univ. of Tromsoe / E-MAIL: johnmcd at fagmed.uit.no> N-9037 Tromsoe /
> Norway /
>
How about all those micro concentrators from amicon and millipore.
Although I tend to believe that you lose a lot of sample in this method,
may be it is worthwhile and quick enough for you to try it out. They have
a whole sleuth of cut off sizes for this.
Raj Shankarappa
bsh at med.pitt.edu